The largest database of trusted experimental protocols

7 protocols using fc blocked

1

Cytokine Stimulation and CyTOF Profiling

Check if the same lab product or an alternative is used in the 5 most similar protocols
Suspension mass cytometry (CyTOF) experiments were performed as previously described25 (link). Briefly, for cytokine stimulation, single-cell samples were stimulated using a pre-mixed cocktail of PMA, ionomycin, and brefeldin A (Biolegend) for 2.5 hours at 37°C. All samples were stained for viability using palladium (Sigma) for 5 minutes at RT and quenched with complete RPMI media. Each batch of up to 10 samples were live-cell barcoded using a 5-choose-3 scheme based on CD45 antibodies tagged with 5 different isotopically enriched metals at room temperature (RT) for 10 minutes. Multiplexed batches were Fc blocked (Invitrogen) and then stained with the antibodies purchased from indicated sources and used at described dilutions (Supplementary Table 4). Chemokine receptor stains were performed first at 37°C for 10 minutes followed by all other surface markers at RT for an additional 20 minutes. Intracellular markers were stained with Cytofix/Cytoperm Kit (BD) as per manufacturer’s protocol. Stained cells were fixed with 1.6% paraformaldehyde (ThermoFisher) in PBS and stored up to 1 week at 4°C. On the day before data acquisition, all cells were stained with rhodium Cell-ID (Fluidigm). All data was acquired using Helios™ at University of Maryland School of Medicine Center for Innovative Biomedical Resources Flow Cytometry and Mass Cytometry Core Facility, Baltimore, Maryland.
+ Open protocol
+ Expand
2

Cytokine Stimulation and CyTOF Profiling

Check if the same lab product or an alternative is used in the 5 most similar protocols
Suspension mass cytometry (CyTOF) experiments were performed as previously described25 (link). Briefly, for cytokine stimulation, single-cell samples were stimulated using a pre-mixed cocktail of PMA, ionomycin, and brefeldin A (Biolegend) for 2.5 hours at 37°C. All samples were stained for viability using palladium (Sigma) for 5 minutes at RT and quenched with complete RPMI media. Each batch of up to 10 samples were live-cell barcoded using a 5-choose-3 scheme based on CD45 antibodies tagged with 5 different isotopically enriched metals at room temperature (RT) for 10 minutes. Multiplexed batches were Fc blocked (Invitrogen) and then stained with the antibodies purchased from indicated sources and used at described dilutions (Supplementary Table 4). Chemokine receptor stains were performed first at 37°C for 10 minutes followed by all other surface markers at RT for an additional 20 minutes. Intracellular markers were stained with Cytofix/Cytoperm Kit (BD) as per manufacturer’s protocol. Stained cells were fixed with 1.6% paraformaldehyde (ThermoFisher) in PBS and stored up to 1 week at 4°C. On the day before data acquisition, all cells were stained with rhodium Cell-ID (Fluidigm). All data was acquired using Helios™ at University of Maryland School of Medicine Center for Innovative Biomedical Resources Flow Cytometry and Mass Cytometry Core Facility, Baltimore, Maryland.
+ Open protocol
+ Expand
3

Immune Cell Profiling in Murine Sepsis

Check if the same lab product or an alternative is used in the 5 most similar protocols
LPS (6 mg/ml) and/or AD-01 (3 mg/kg) or equal volume sterile PBS was injected via intraperitoneal injection into C57BL/J6 wild-typeor Fkbpl +/-mice. Following a 3 h incubation mice were culled by cervical dislocation and peritoneal lavage was performed with 3 ml of PBS containing 3% BSA. Lavage fluid was harvested and cells were Fc blocked (Invitrogen, Ireland) and stained on ice for 30 min (Table 3) and gated as outlined in Supplementary Figure 2.
Compensation was determined by single staining alone and no positive staining detected from IgG controls. Analysis was conducted using FlowJo Version 2 (Flowjo, USA).
+ Open protocol
+ Expand
4

Characterization of Airway Immune Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
Immune cells were harvested from the airways via bronchoalveolar lavage (BAL). Briefly, mice were cannulated via a small tracheal incision and the lungs were flushed with 1 mL of sterile PBS. The collected BAL fluids were centrifuged at 1,500 rpm for 5 min at 4°C, and total cells were prepared for flow cytometry staining to determine the number and types of cells. Fc-blocked (1 μg/mL; eBiosciences) BALF cells were stained with anti-mouse SiglecF-PE (0.3 μg/mL; BD Pharmingen), CD11c-APC (0.3 μg/mL; eBiosciences), CD11b-PerCP (0.3 μg/mL; BioLegend), CD19-PECy5 (0.8 μg/mL; eBiosciences), Ly6G-PECy7 (0.8 μg/mL; BioLegend), Ly6C-APC-Cy7 (0.8 μg/mL; BD Pharmingen), and TCRβ-Pacific Blue (0.3 μg/mL; BioLegend). All samples were analyzed on a Becton-Dickinson LSR-II/Fortessa flow cytometer (BD Biosciences, San Diego, CA, USA) and analyzed by using FlowJo software (Tree Star Inc.).
+ Open protocol
+ Expand
5

Comprehensive Immune Cell Profiling

Check if the same lab product or an alternative is used in the 5 most similar protocols
Single cell suspensions were washed with PBS and 1.0 × 106 cells/tube were FC blocked (eBioscience) and stained extracellularly for 30 min at 4 °C. Cells were then washed and prepared for intracellular staining using the FoxP3 Intracellular Staining Kit (R and D systems). Intracellular staining was performed at 4 °C for 60 min. Antibodies CD79A-APC, CD27-FITC, IgD-PE, CD4-APC, CD45RO-PE, CD8-FITC, and CD3-APC were purchased from eBioscience.
+ Open protocol
+ Expand
6

Adoptive Transfer of OT-1 CD8+ T cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
OT-1 CD8+ T cells (in C57BL/6 background) were isolated from the spleens of CD45.2 mice and 1 × 106 cells were transplanted into C57BL/6 CD45.1 hosts. 24 h later, transplanted and control mice were injected i.p. with 100 μg OVA, 100 μg anti-CD40, and 50 μg Poly (I:C). Peripheral blood was acquired through submandibular bleed every 7 days thereafter. Peripheral blood was RBC lysed (ACK Buffer, Fisher) and Fc-blocked (eBioscience) and WBCs were stained with CD45.1, CD45.2, and CD8 (eBioscience) and KB-OVA tetramer (MLB International) in PBS supplemented with 0.5% Bovine Serum Albumin for 30 min on ice. Cells were washed twice and data was collected on an LSRII (Beckman Dickinson) and analyzed with FlowJo 9.6 (TreeStar). Cells were gated on strict forward and side scatter parameters, to ensure single cell analysis. Additionally, the DAPI- population was used for viable cells. Finally, cells were separated by CD45.1 or CD45.2 positivity, and assessed for CD8/OVA-tetramer expression.
+ Open protocol
+ Expand
7

BAL Immune Cell Profiling by Flow Cytometry

Check if the same lab product or an alternative is used in the 5 most similar protocols
Flow cytometry was used to determine the number and types of cells present in the BAL fluid. Fc-blocked (1 µg/ml; eBiosciences) BAL cells were stained with anti-mouse SiglecF-phycoerythrin (PE; 1 µg/ml; BD Pharmingen), CD45- allophycocyanin (APC; 1 µg/ml; eBiosciences), CD3-PECy5 (1 µg/ml; eBiosciences), CD19-PECy5 (1 µg/ml; eBiosciences), CD11c-PECy7 (1 µg/ml; eBiosciences), MHCII-APC-efluor780 (1 µg/ml; eBiosciences), CD11b-V450 (1 µg/ml; BD Pharmingen), Ly6C-FITC 1 µg/ml; BD Pharmingen) and Ly6G-AlexaFluor700 (1 µg/ml; BD Pharmingen). Fixable Viability Dye eFluor® 506 (eBiosciences) was added to exclude dead cells from the analysis.
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!